The invention concerns a method for controlling the position or the motion of objects in field cages and a device for performing such a method.
In numerous biological, medical, pharmacological and physical or chemical engineering processes one is interested in holding microscopic particles, especially biological cells or their constituents, latex particles or other microbeads, as precisely as possible in position or moving them along a certain path. One known method of retaining biological cells is that of growing them on a solid substrate, which can then be set with the required accuracy; for example, with reference to a means of measurement. The drawback of this technique is that of creating a mechanical contact with the substrate surface and the generally difficult separation of this contact, after performing a measurement. Furthermore, this technique is restricted to retaining objects and unsuitable for moving them along a certain path.
Another known technique is that of positioning objects in free fluids by optical means. With so-called laser tweezers, it is possible to hold particles in free solutions with micrometer accuracy or to shift them in a predetermined manner (see A. Ashkin et al. xe2x80x9cObservation of a single-beam gradient force optical trap for dielectric particlesxe2x80x9d in Optics Lett., vol. 11, p 288 (1986)). This so-called optical trapping presents drawbacks, because the particle within the trapping laser beam is subjected to thermal collisions and thus slight, random shifts in position. Furthermore, optical measurements on retained particles are limited in scope because of interference with the laser beam. The latter restriction is avoided in the manipulation of particles in electric microfield cages where the objects can be retained or moved by polarization forces (see G. Fuhr et al. xe2x80x9cRadio-frequency microtools for particle and live cell manipulationxe2x80x9d in Naturwissenschaften, vol. 81, p 528 (1994), or: DE-OS 195 00 660)). Nevertheless, here too there are thermally related shifts in the position of the retained or moved particles that can make high-resolution or high-sensitivity measurements as in correlation spectroscopy (see M. Eigen et al. xe2x80x9cSorting single molecules, application to diagnostics and evolutionary biotechnologyxe2x80x9d in Proc. Nat. Acad. Sci. USA, volume 91, p 5740 (1994)), difficult or even impossible.
It is a known principle where the particle to be retained is made to vibrate in the required position to enable locally triggered measurement with reduced interference from thermal collisions. For this purpose the retained particle in the required position is made to vibrate by sound (especially ultrasonic sound) or periodic movements of solution so that there is a certain periodic movement at the required position. The correspondingly modulated measurement signal shows an improved signal/noise ratio.
This vibration technique exhibits the following disadvantages however. The forces for creating the vibration act indirectly, through the surrounding fluid, on the retained particle. So not only the particle but also the surrounding fluid is moved, thus reducing the reproducibility of particle positioning. Moreover, unwanted rotary motion, difficult to control, can appear as a result of this indirect action of forces. Certain applications where motion of the surrounding fluid is undesired cannot be implemented. Finally, mechanical stress can occur on the retained particle, which is a special problem with living biological objects.
The known techniques do not allow one to influence the positioning of a particle along a required path. But there is also a requirement for control of motion beyond what is possible with the above mentioned vibration technique.
Accordingly, it is the object of the invention to provide an improved method of positioning objects with electric field cages and to propose a device for implementing the said method, extending the usability of the above mentioned vibration technique and creating new possibilities of control.
This object is solved by a method with the features of patent claim 1 and a device with the features of patent claim 9. Advantageous embodiments of the invention are defined in the dependent claims.
The invention is based on the idea of departing from the use of forces acting indirectly through motion of the fluid when positioning by superimposing vibration or using other means to control motion, and instead focuses on moving the object a periodically or periodically through the effect of forces that act directly on the retained object but not on the surrounding fluid (suspension, solution). For this purpose electric potentials on electrodes of a multi-electrode configuration are used to create an open or closed field cage. Such potentials are superimposed with drive or control potentials so that the position of the field minimum, where the particular object is to be found, changes in relation to a required object position. The required object position can be either a certain location within a closed field cage or a path within an open field cage (duct structure or the like). A periodic change of position means that the retained particle is conducted periodically on a path around the required, predetermined object position. The shape of this superimposed trajectory can be chosen as desired and can include a vibratory motion in relation to one, two or three spatial axes, circular, elliptical, essentially rectangular, or even in more complex paths. The time pattern of the spatially periodic motion can itself exhibit periodic modulation. An aperiodic change of position means that the retained particle is led away from a predetermined object position on a certain path.
If the required object position is formed by a path within an open field cage, the moving of the object along the required path can be produced by appropriate selection of the driving of the electrodes with base potentials and/or by a fluid flow.
A device according to the invention possesses an electrode arrangement that can be driven by a combination of high-frequency generators and a switching device. The switching device is adapted to superimpose a drive potential on the potential (basic potential) of one or more electrodes so that the amplitude, the frequency and/or the phase of the resulting total potential alters.
The invention produces the following advantages, especially in the positioning of objects that are freely supported with surrounding fluids in field cages.
The positioning or moving of the object is performed without mechanical contact. The objects are not exposed to any disturbing mechanical stress, so in particular live biological cells and the like are not damaged. There are no restrictions at all with regard to further manipulation or measurement of the retained objects. Common optical methods of measurement can be employed while avoiding interference problems. Motion of the objects about the required location or on the required path can be created in complex reproducible manner, differing from harmonic vibratory processes. The superimposed motion of the objects can be controlled or programmed in a predetermined way. There is a unique relation between the form of the drive potentials and the object motions that are produced, so no feedback or observation of the moved object is necessary for control purposes. Finally, the invention can be used with any electrode arrangements to create field cages, independently of the concrete electrode form or configuration.
Preferred uses for the methods and devices according to the invention are correlation spectroscopy, especially for detecting fluorescent molecules on the surface of submicrometer or micrometer particles and/or cells, pharmacological and medical diagnostic applications and/or evolution biotechnology. Especially suitable as means of detection are the method of fluorescence correlation spectroscopy (WO 94/16313) as well as other, particularly confocal fluorescence techniques, as proposed in the publication WO 96/13744 and the European patent application 96116373.0. This latter application suggests a method for analyzing samples by repeated measurement of the number of photons per predetermined time interval of the electromagnetic radiation, in particular light, that is emitted, scattered and/or reflected by the particles in the sample, and determination of the distribution of the number of photons in the particular time intervals, whereby the distribution of the molecular intensity of the particles obtained from the distribution of the number of photons.